U.S. patent application number 10/165168 was filed with the patent office on 2003-01-02 for light modulated microarray reader and methods relating thereto.
Invention is credited to Lane, Pierre M., MacAulay, Calum E..
Application Number | 20030002040 10/165168 |
Document ID | / |
Family ID | 23142861 |
Filed Date | 2003-01-02 |
United States Patent
Application |
20030002040 |
Kind Code |
A1 |
MacAulay, Calum E. ; et
al. |
January 2, 2003 |
Light modulated microarray reader and methods relating thereto
Abstract
Microarray readers and methods that compensate for target spots
that are too dim or too bright for the microarray reader to
accurately measure. The readers adjust the amount of light directed
at or received from specific non-acceptable target spots, such that
dim spots receive more excitation light and overly bright spots
receive less. This increases or decreases, respectively, their
measured brightness, which in turn effectively increases the range
over which a microarray reader can accurately measure the spots,
and can also improve the signal-to-noise ratio and other aspects of
the measurements.
Inventors: |
MacAulay, Calum E.;
(Vancouver, CA) ; Lane, Pierre M.; (Vancouver,
CA) |
Correspondence
Address: |
GRAYBEAL, JACKSON, HALEY LLP
155 - 108TH AVENUE NE
SUITE 350
BELLEVUE
WA
98004-5901
US
|
Family ID: |
23142861 |
Appl. No.: |
10/165168 |
Filed: |
June 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60296635 |
Jun 6, 2001 |
|
|
|
Current U.S.
Class: |
356/317 ;
250/458.1; 356/417 |
Current CPC
Class: |
G01N 21/253 20130101;
G01N 21/6452 20130101; G02B 26/02 20130101; G02B 27/021
20130101 |
Class at
Publication: |
356/317 ;
250/458.1; 356/417 |
International
Class: |
G01N 021/64 |
Claims
What is claimed is:
1. An automated method of reading a microarray comprising, a)
providing an initial representation of a microarray comprising a
plurality of target spots illuminated by illumination light having
a designated intensity; b) determining from the initial
representation whether at least one of the target spots has an
emanating light intensity that is not between selected upper and
lower threshold values, and designating such target spot a
non-acceptable target spot; and, c) modulating the designated
intensity of the illumination light via an automated upstream
selective light modulator located in an illumination light path
substantially at a conjugate image plane of the sample to provide a
modulated illumination light and an adjusted target spot that
emanates an adjusted light intensity between the selected upper and
lower threshold values.
2. The method of claim 1 wherein the method further comprises
measuring the amount of modulation of the designated intensity of
the illumination light and measuring the adjusted light intensity,
then correlating the amount of modulation with the adjusted light
intensity to provide a measure of the actual signal strength of the
target spot.
3. The method of claim 2 wherein the method further comprises
determining an amount of a probe located at the adjusted target
spot from the measure of the actual signal strength of the target
spot.
4. The method of claim 2 wherein the method is implemented
according to a formula: SS(x,y)=K*CCDS(x,y)/II(x,y) where, SS(x,y)
is the actual signal strength of the target spot, K is a constant
for the system, (CCDS(x,y)) is the adjusted light intensity, and
(II(x,y)) is the modulated illumination light.
5. The method of claim 2 wherein the method further comprises
detecting a fluorescent target spot.
6. The method of claim 5 wherein the method is implemented
according to a formula:
SS(x,y)=K*PB(II(x,y),fluoro)*CCDS(x,y)/II(x,y) where, SS(x,y) is
the actual signal strength of the target spot, K is a constant for
the system, PB(II(x,y),fluoro) is a photobleaching function based
on illumination energy/intensity and a fluorophore being excited,
(CCDS(x,y)) is the adjusted light intensity, and (II(x,y)) is the
modulated illumination light.
7. The method of claim 5 wherein the method is implemented
according to a formula:
SS(x,y)=K*PB(II(x,y),fluoro,x,y)*CCDS(x,y)/II(x,y) where, SS(x,y)
is the actual signal strength of the target spot, K is a constant
for the system, PB(II(x,y),fluoro,x,y) is a photobleaching function
based on illumination energy/intensity, a fluorophore being
excited, and a spatial variation term, (CCDS(x,y)) is the adjusted
light intensity, and (II(x,y)) is the modulated illumination
light.
8. The method of claim 2 wherein the modulated illumination light
is modulated by changing its illumination intensity.
9. The method of claim 2 wherein the modulated illumination light
is modulated by changing its duration of illuminating the target
spot.
10. The method of claim 2 wherein the initial representation
comprises a precompiled map of expected data for the target spots
of the microarray.
11. The method of claim 2 wherein the initial representation
comprises an initial image of the plurality of target spots
illuminated by the illumination light having the designated
intensity and taken by a same microarray reader that implements the
determining, modulating, measuring and correlating.
12. The method of claim 11 wherein the initial image is taken
substantially immediately before the determining, modulating,
measuring and correlating are implemented.
13. The method of claim 2 wherein the method further comprises
repeating of the determining, modulating, measuring and correlating
using the measure of the actual signal strength as the initial
representation.
14. The method of claim 2 wherein the method further comprises
selecting a probe such that the modulation is linearly related to
the adjusted light intensity.
15. The method of claim 2 wherein the method is implemented using a
microarray reader comprising the upstream selective light
modulator, and a light detector disposed downstream from the
microarray in a detection light path substantially at a conjugate
image plane of the sample, wherein the selective light modulator
and the light detector are operably connected to at least one
controller containing computer-implemented programming that
controls transmissive characteristics of the upstream selective
light modulator and that compiles the modulated illumination light
and the adjusted light intensity, and wherein the controller
spatially varies the transmissive characteristics of the selective
light modulator to vary the modulated illumination light impinging
on the non-acceptable target spots of the microarray such that
light emanating from the non-acceptable target spots is between the
threshold levels.
16. The method of claim 15 wherein the upstream selective light
modulator comprises a digital micromirror device.
17. The method of claim 15 wherein the detector comprises a charge
coupled device.
18. An automated method of reading a microarray comprising, a)
providing an initial representation of a microarray comprising a
plurality of target spots illuminated by illumination light having
a designated intensity; b) determining from the initial
representation whether at least one of the target spots has an
emanating light intensity that is not between selected upper and
lower threshold values, and designating at least one of such target
spots a non-acceptable target spot; and, c) modulating the
emanating light intensity via an automated downstream selective
light modulator located in a detection light path substantially at
a conjugate image plane of the sample to provide a modulated
detection light comprising an adjusted emanating light intensity
that is between the selected upper and lower threshold values.
19. The method of claim 18 wherein the method further comprises
measuring the amount of modulation of the detection light and
measuring the modulated detection light, then correlating the
amount of modulation with the modulated detection light to provide
a measure of the actual signal strength of the target spot.
20. The method of claim 19 wherein the method further comprises
determining an amount of a probe located at the non-acceptable
target spot from the measure of the actual signal strength of the
target spot.
21. The method of claim 19 wherein the method is implemented
according to a formula: SS(x,y)=K*CCDS(x,y)/II(x,y) where, SS(x,y)
is the actual signal strength of the target spot, K is a constant
for the system, (CCDS(x,y)) is the adjusted light intensity, and
(II(x,y)) is the modulated illumination light.
22. The method of claim 19 wherein the method further comprises
detecting a fluorescent target spot.
23. The method of claim 22 wherein is implemented according to a
formula: SS(x,y)=K*PB(II(x,y),fluoro)*CCDS(x,y)/II(x,y) where,
SS(x,y) is the actual signal strength of the target spot, K is a
constant for the system, PB(II(x,y),fluoro) is a photobleaching
function based on illumination energy/intensity and a fluorophore
being excited, (CCDS(x,y)) is the adjusted light intensity, and
(II(x,y)) is the modulated illumination light.
24. The method of claim 22 wherein the method is implemented
according to a formula:
SS(x,y)=K*PB(II(x,y),fluoro,x,y)*CCDS(x,y)/II(x,y) where, SS(x,y)
is the actual signal strength of the target spot, K is a constant
for the system, PB(II(x,y),fluoro,x,y) is a photobleaching function
based on illumination energy/intensity, a fluorophore being
excited, and a spatial variation term, (CCDS(x,y)) is the adjusted
light intensity, and (II(x,y)) is the modulated illumination
light.
25. The method of claim 19 wherein the initial representation
comprises a precompiled map of expected data for the target spots
of the microarray.
26. The method of claim 19 wherein the initial representation
comprises an initial image of the plurality of target spots
illuminated by the illumination light having the designated
intensity and taken by a same microarray reader that implements the
determining, modulating, measuring and correlating.
27. The method of claim 26 wherein the initial image is taken
substantially immediately before the determining, modulating,
measuring and correlating are implemented.
28. The method of claim 19 wherein the method further comprises
repeating of the determining, modulating, measuring and correlating
using the measure of the actual signal strength as the initial
representation.
29. The method of claim 19 wherein the method further comprises
selecting a probe such that the modulation is linearly related to
the adjusted light intensity.
30. The method of claim 19 wherein the method is implemented using
a microarray reader comprising the selective light modulator, and a
light detector disposed in a detection light path substantially at
a conjugate image plane of the sample and downstream from the
microarray and the downstream selective light modulator, wherein
the selective light modulator and the light detector are operably
connected to at least one controller containing
computer-implemented programming that controls transmissive
characteristics of the downstream selective light modulator and
that compiles the modulated detection light and the adjusted light
intensity, and wherein the controller selectively varies the
transmissive characteristics of the selective light modulator to
vary the modulated detection light impinging on the non-acceptable
target spots of the microarray such that light received at the
detector is between the threshold levels.
31. The method of claim 30 wherein the downstream selective light
modulator comprises a digital micromirror device.
32. The method of claim 30 wherein the detector comprises a charge
coupled device.
33. A microarray reader comprising an automated upstream selective
light modulator located upstream of a microarray in an illumination
light path substantially at a conjugate image plane of the sample,
and a light detector disposed downstream from the microarray in a
detection light path substantially at a conjugate image plane of
the sample, wherein the selective light modulator and the light
detector are operably connected to at least one controller
containing computer-implemented programming that controls
transmissive characteristics of the upstream selective light
modulator and that compiles an amount of modulated illumination
light when the upstream selective light modulator is modulated and
an adjusted light intensity emanating from a target spot on a
microarray receiving the modulated illumination light, and wherein
the controller selectively varies the transmissive characteristics
of the selective light modulator to vary the modulated illumination
light impinging on at least one non-acceptable target spot of the
microarray such that light emanating from the at least one
non-acceptable target spot is between selected threshold
levels.
34. The microarray reader of claim 33 wherein the controller
further comprises computer-implemented programming that controls
measuring the amount of modulation of the illumination light and
controls measuring the adjusted light intensity, then correlates
the amount of modulation with the adjusted light intensity to
provide a measure of the actual signal strength of the target
spot.
35. The microarray reader of claim 33 wherein the controller
further comprises computer-implemented programming that determines
an amount of a probe located at the at least one non-acceptable
target spot from the measure of the actual signal strength of the
target spot.
36. The microarray reader of claim 34 wherein the controller
further comprises computer-implemented programming comprising the
formula: SS(x,y)=K*CCDS(x,y)/II(x,y) where, SS(x,y) is the actual
signal strength of the target spot, K is a constant for the system,
(CCDS(x,y)) is the adjusted light intensity, and (II(x,y)) is the
modulated illumination light.
37. The microarray reader of claim 34 wherein the controller
further comprises computer-implemented programming comprising the
formula: SS(x,y)=K*PB(II(x,y),fluoro)*CCDS(x,y)/II(x,y) where,
SS(x,y) is the actual signal strength of the target spot, K is a
constant for the system, PB(II(x,y),fluoro) is a photobleaching
function based on illumination energy/intensity and a fluorophore
being excited, (CCDS(x,y)) is the adjusted light intensity, and
(II(x,y)) is the modulated illumination light.
38. The microarray reader of claim 34 wherein the controller
further comprises computer-implemented programming comprising the
formula: SS(x,y)=K*PB(II(x,y),fluoro,x,y)*CCDS(x,y)/II(x,y) where,
SS(x,y) is the actual signal strength of the target spot, K is a
constant for the system, PB(II(x,y),fluoro,x,y) is a photobleaching
function based on illumination energy/intensity, a fluorophore
being excited, and a spatial variation term, (CCDS(x,y)) is the
adjusted light intensity, and (II(x,y)) is the modulated
illumination light.
39. The microarray reader of claim 34 wherein the controller
further comprises computer-implemented programming comprising a
precompiled map of expected data for the target spots of the
microarray.
40. The microarray reader of claim 34 wherein the upstream
selective light modulator comprises a digital micromirror
device.
41. The microarray reader of claim 34 wherein the detector
comprises a charge coupled device.
42. A microarray reader comprising an automated downstream
selective light modulator located downstream of a microarray in a
detection light path substantially at a conjugate image plane of
the sample, and a light detector disposed in the detection light
path substantially at a conjugate image plane of the sample and
downstream from the downstream selective light modulator and the
microarray, wherein the downstream selective light modulator and
the light detector are operably connected to at least one
controller containing computer-implemented programming that
controls transmissive characteristics of the downstream selective
light modulator and that compiles an amount of modulated detection
light when the downstream selective light modulator is modulated
and an adjusted light intensity received by the detector, and
wherein the controller selectively varies the transmissive
characteristics of the downstream selective light modulator to vary
the modulated detection light emanating from at least one
non-acceptable target spot of the microarray such that light
received at the detector from the at least one non-acceptable
target spot is between selected threshold levels.
43. The microarray reader of claim 42 wherein the controller
further comprises computer-implemented programming that controls
measuring the amount of modulation of the detection light and
controls measuring the adjusted light intensity, then correlates
the amount of modulation with the adjusted light intensity to
provide a measure of the actual signal strength of the target
spot.
44. The microarray reader of claim 43 wherein the controller
further comprises computer-implemented programming that determines
an amount of a probe located at the at least one non-acceptable
target spot from the measure of the actual signal strength of the
target spot.
45. The microarray reader of claim 43 wherein the controller
further comprises computer-implemented programming comprising the
formula: SS(x,y)=K*CCDS(x,y)/II(x,y) where, SS(x,y) is the actual
signal strength of the target spot, K is a constant for the system,
(CCDS(x,y)) is the adjusted light intensity, and (II(x,y)) is the
modulated illumination light.
46. The microarray reader of claim 43 wherein the controller
further comprises computer-implemented programming comprising the
formula: SS(x,y)=K*PB(II(x,y),fluoro)*CCDS(x,y)/II(x,y) where,
SS(x,y) is the actual signal strength of the target spot, K is a
constant for the system, PB(II(x,y),fluoro) is a photobleaching
function based on illumination energy/intensity and a fluorophore
being excited, (CCDS(x,y)) is the adjusted light intensity, and
(II(x,y)) is the modulated illumination light.
47. The microarray reader of claim 43 wherein the controller
further comprises computer-implemented programming comprising the
formula: SS(x,y)=K*PB(II(x,y),fluoro,x,y)*CCDS(x,y)/II(x,y) where,
SS(x,y) is the actual signal strength of the target spot, K is a
constant for the system, PB(II(x,y),fluoro,x,y) is a photobleaching
function based on illumination energy/intensity, a fluorophore
being excited, and a spatial variation term, (CCDS(x,y)) is the
adjusted light intensity, and (II(x,y)) is the modulated
illumination light.
48. The microarray reader of claim 43 wherein the controller
further comprises computer-implemented programming comprising a
precompiled map of expected data for the target spots of the
microarray.
49. The microarray reader of claim 43 wherein the downstream
selective light modulator comprises a digital micromirror
device.
50. The microarray reader of claim 43 wherein the detector
comprises a charge coupled device.
51. An automated method of reading a microarray comprising, a)
providing an initial representation of a microarray comprising a
plurality of target spots illuminated by illumination light having
a designated intensity; b) determining from the initial
representation whether at least one of the target spots has an
emanating light intensity that is not between selected upper and
lower threshold values, and designating at least one of such target
spots as a non-acceptable target spot; c) selectively illuminating
the non-acceptable target spot via selectively transmitting light
to the microarray using a first automated upstream selective light
modulator located in an illumination light path substantially at a
conjugate image plane of the sample; and, d) modulating the
designated intensity of the illumination light via a second
automated upstream selective light modulator located in the
illumination light path substantially at a conjugate image plane of
an aperture diaphragm of the objective lens, to provide a modulated
illumination light and an adjusted target spot that emanates an
adjusted light intensity between the selected upper and lower
threshold values.
52. The method of claim 51 wherein the method further comprises
measuring the amount of modulation of the designated intensity of
the illumination light and measuring the adjusted light intensity,
then correlating the amount of modulation with the adjusted light
intensity to provide a measure of the actual signal strength of the
target spot.
53. The method of claim 52 wherein the method further comprises
also modulating the designated intensity of the illumination light
via the first automated upstream selective light modulator located
in the illumination light path substantially at the conjugate image
plane of the sample.
54. The method of claim 52 wherein the method further comprises
determining an amount of a probe located at the adjusted target
spot from the measure of the actual signal strength of the target
spot.
55. An automated method of reading a microarray comprising, a)
providing an initial representation of a microarray comprising a
plurality of target spots illuminated by illumination light having
a designated intensity; b) determining from the initial
representation whether at least one of the target spots has an
emanating light intensity that is not between selected upper and
lower threshold values, and designating at least one of such target
spots as a non-acceptable target spot; c) selectively detecting
light from the non-acceptable target spot via selectively
transmitting light from the microarray using a first automated
downstream selective light modulator located in a detection light
path substantially at a conjugate image plane of the sample; and,
d) modulating the emanating light intensity via a second automated
downstream selective light modulator located in a detection light
path substantially at a conjugate image plane of an aperture
diaphragm of the objective lens, to provide a modulated detection
light comprising an adjusted emanating light intensity between the
selected upper and lower threshold values.
56. The method of claim 55 wherein the method further comprises
measuring the amount of modulation of the emanating light intensity
and measuring the modulated detection light, them correlating the
amount of modulation with the modulation detection light to provide
a measure of the actual signal strength of the target spot.
57. The method of claim 56 wherein the method further comprises
also modulating the emanating light intensity of the detection
light via the first automated downstream selective light modulator
located in the detection light path substantially at the conjugate
image plane of the sample.
58. The method of claim 56 wherein the method further comprises
determining an amount of a probe located at the adjusted target
spot from the measure of the actual signal strength of the target
spot.
59. A microarray reader comprising a first automated upstream
selective light modulator located upstream of a microarray in an
illumination light path substantially at a conjugate image plane of
the sample, a second automated upstream selective light modulator
located upstream of the microarray in the illumination light path
substantially at a conjugate image plane of an aperture diaphragm
of the objective lens, and a light detector disposed downstream
from the microarray in a detection light path substantially at a
conjugate image plane of the sample, wherein the first and second
selective light modulators and the light detector are operably
connected to at least one controller containing
computer-implemented programming that controls transmissive
characteristics of the first and second upstream selective light
modulators and that compiles an amount of modulated illumination
light when the second upstream selective light modulator is
modulated and an adjusted light intensity emanating from a target
spot on a microarray receiving the modulated illumination light,
and wherein the controller selectively varies the transmissive
characteristics of the second selective light modulator to vary the
modulated illumination light impinging on at least one
non-acceptable target spot of the microarray such that light
emanating from the at least one non-acceptable target spot is
between selected threshold levels.
60. The microarray reader of claim 59 wherein the controller
further comprises computer-implemented programming that controls
measuring the amount of modulation of the illumination light and
controls measuring the adjusted light intensity, then correlates
the amount of modulation with the adjusted light intensity to
provide a measure of the actual signal strength of the target
spot.
61. The microarray reader of claim 60 wherein the controller
further comprises computer-implemented programming that determines
an amount of a probe located at the at least one non-acceptable
target spot from the measure of the actual signal strength of the
target spot.
62. A microarray reader comprising a first automated downstream
selective light modulator located downstream of a microarray in a
detection light path substantially at a conjugate image plane of
the sample, a second automated downstream selective light modulator
located downstream of the microarray in the illumination light path
substantially at a conjugate image plane of an aperture diaphragm
of the objective lens, and a light detector disposed in a detection
light path substantially at a conjugate image plane of the sample
and downstream from the first and second downstream selective light
modulators and the microarray, wherein the first and second
selective light modulators and the light detector are operably
connected to at least one controller containing
computer-implemented programming that controls transmissive
characteristics of the first and second downstream selective light
modulator and that compiles an amount of modulated detection light
when the second downstream selective light modulator is modulated
and an adjusted emanating light intensity received by the detector,
and wherein the controller selectively varies the transmissive
characteristics of the second downstream selective light modulator
to vary the modulated detection light emanating from at least one
non-acceptable target spot of the microarray such that light
received at the detector from the at least one non-acceptable
target spot is between selected threshold levels.
63. The microarray reader of claim 62 wherein the controller
further comprises computer-implemented programming that controls
measuring the amount of modulation of the detection light and
controls measuring the adjusted emanating light intensity, then
correlates the amount of modulation with the adjusted emanating
light intensity to provide a measure of the actual signal strength
of the target spot.
64. The microarray reader of claim 63 wherein the controller
further comprises computer-implemented programming that determines
an amount of a probe located at the at least one non-acceptable
target spot from the measure of the actual signal strength of the
target spot.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority from U.S.
provisional patent application No. 60/296,635, filed Jun. 6,
2001.
BACKGROUND
[0002] "Microarrays" are devices used in biotechnology and other
science research, and can be made by putting a large number of tiny
droplets of DNA (or other target such as cDNA or proteins) on a
glass slide. Short pieces of DNA, called probes, are then applied
to the DNAs on the slide. Typically, the probes are fluorescent, so
they light up when short wavelength light is shone on them (the
probes can also be labeled with other substances to reflect or
otherwise emanate light when they are scanned). Microarrays can be
used, for example, to study how large numbers of genes interact
with each other (genes are made of DNA), or how a cell is able to
simultaneously control vast numbers of genes.
[0003] The probes stick to the microarray wherever the probes find
target stretches of DNA called complementary DNA strands. The
microarrays are then put into a scanning microarray reader that
measures the brightness of each fluorescent dot: the brighter the
dot, the more probe (and thus the more target DNA or other
biological material) is present. This can indicate, for example,
how active the target is, or where it is on the slide.
[0004] Microarrays can be used, for example, to study genomic
content, how large numbers of genes interact with each other (genes
are made of DNA), or how a cell is able to simultaneously control
vast numbers of genes (expression patterns). Different types of
microarrays include, but are not limited to, cDNA arrays,
oligonucleotide arrays and protein arrays.
[0005] Some general concepts about microarrays, and the microarray
readers that measure the dots, are discussed in more scientific
terms in the following paragraphs.
[0006] Fluorescence based microarray readers suffer from limited
dynamic range with respect to the possible intensities of target
spots in, for example, cDNA expression microarrays. Systems which
use scanning spots and photomultiplier tubes (PMTs) for detection
are reported to possibly have a .about.10.sup.6 dynamic range but
may have a 10.sup.4 to 10.sup.5 dynamic range in practice. Most
charge couple device (CCD) imaging microarray readers have about a
.about.10.sup.3 dynamic range (12 bit-digitization, 4096 levels,
40,000-1,000,000 electron well depths, 200:1 up to 1000:1 possible
signal-to-noise ratio).
[0007] The intensity of a fluorescent target spot on a microarray
is a function of factors such as how long the target spot is
sampled (data sampling time, which can be dwell time for scanning
spot systems and integration time for CCD imaging systems), the
intensity of the illumination (illumination intensity), the
sensitivity of the detector (quantum efficiency, signal transducer,
or measurement sensitivity), and the accuracy of digitization
(pulse counting or voltage digitization). Coordination and control
of these factors is difficult, so measuring spot intensity over a
wide range is difficult.
[0008] Accordingly, there has gone unmet a need for improved
methods of precisely measuring the brightness of the target spots
on a microarray over a wide range of target spot intensity. The
present invention provides this and other advantages.
SUMMARY
[0009] Target spots on a microarray that are too dim or too bright
for the microarray reader to accurately measure are a problem, for
example because they fall outside of certain threshold levels so
the microarray reader cannot accurately measure them, or because
target spots that are too bright can also hinder the measurement of
neighboring target spots due to glare or other interference.
Typically, the intensity of light emanating from target spots is
proportional to the amount of light shown or incident on the target
spots; the more light that is incident on the target spot
(excitation or illumination light), the brighter the light coming
from the target spot. The present invention takes advantage of this
and adjusts the amount of light directed at specific non-acceptable
target spots (for example, those spots which fall outside the
dynamic range of the system in use), such that dim spots receive
more excitation light and overly bright spots receive less.
[0010] Similarly, in conjunction with or instead of such actions,
the present invention adjusts the amount of light received from
specific non-acceptable target spots, such that the detector
receives more light from dim spots and less light from overly
bright spots. This increases or decreases, respectively, their
measured brightness, which in turn effectively increases the range
over which a microarray reader can accurately measure the spots,
and can also improve the signal-to-noise ratio and other aspects of
the measurements. In some embodiments, the present invention can
increase the range of the microarray reader by up to about 1000
times or more, and improve the signal-to-noise ratio for target
spots up to about 16 times or more.
[0011] In one aspect, the present invention provides automated
methods of reading a microarray comprising, a) providing an initial
representation of a microarray comprising a plurality of target
spots illuminated by illumination light having a designated
intensity; b) determining from the initial representation whether
at least one of the target spots has an emanating light intensity
that can be not between selected upper and lower threshold values,
and designating such target spot a non-acceptable target spot; and,
c) modulating the designated intensity of the illumination light
via an automated upstream selective light modulator located in an
illumination light path substantially at a conjugate image plane of
the sample to provide a modulated illumination light and an
adjusted target spot that emanates an adjusted light intensity
between the selected upper and lower threshold values.
[0012] In some embodiments, the methods further comprise measuring
the amount of modulation of the designated intensity of the
illumination light and measuring the adjusted light intensity, then
correlating the amount of modulation with the adjusted light
intensity to provide a measure of the actual signal strength of the
target spot. In this and other embodiments of the invention (unless
expressly stated otherwise or clear from the context), all
embodiments, aspects, features, etc., of the present invention can
be mixed and matched, combined and permuted in any desired manner.
The methods can further comprise determining an amount of a probe
located at the adjusted target spot from the measure of the actual
signal strength of the target spot. The methods are suitable for
detecting any light emanating spot, such as reflective, fluorescent
or other light.
[0013] The methods can be implemented according to various
formulae. Such formulae include:
SS(x,y)=K*CCDS(x,y)/II(x,y) (1)
[0014] where,
[0015] SS(x,y) can be the actual signal strength of the target
spot,
[0016] K can be a constant for the system,
[0017] (CCDS(x,y)) can be the adjusted light intensity, and
[0018] (II(x,y)) can be the modulated illumination light.
SS(x,y)=K*PB(II(x,y),fluoro)*CCDS(x,y)/II(x,y) (2)
[0019] where,
[0020] SS(x,y) can be the actual signal strength of the target
spot,
[0021] K can be a constant for the system,
[0022] PB(II(x,y),fluoro) can be a photobleaching function based on
illumination energy/intensity and a fluorophore being excited,
[0023] (CCDS(x,y)) can be the adjusted light intensity, and
[0024] (II(x,y)) can be the modulated illumination light.
SS(x,y)=K*PB(II(x,y),fluoro,x,y)*CCDS(x,y)/II(x,y) (3)
[0025] where,
[0026] SS(x,y) can be the actual signal strength of the target
spot,
[0027] K can be a constant for the system,
[0028] PB(II(x,y),fluoro,x,y) can be a photobleaching function
based on illumination energy/intensity, a fluorophore being
excited, and a spatial variation term,
[0029] (CCDS(x,y)) can be the adjusted light intensity, and
[0030] (II(x,y)) can be the modulated illumination light.
[0031] The modulated illumination light can be modulated by
changing its illumination intensity, by changing its duration of
illuminating the target spot, or otherwise as desired. The initial
representation can comprise a precompiled map of expected data for
the target spots of the microarray, or an initial image of the
plurality of target spots illuminated by the illumination light
having the designated intensity, typically taken by a same
microarray reader that implements other elements of the methods.
The initial image can be taken substantially immediately before the
determining, modulating, measuring and correlating are
implemented.
[0032] The methods can further comprise repeating any desired
element, such as the determining, modulating, measuring and
correlating in an iterative fashion, for example using the measure
of the actual signal strength as the initial representation. The
probe can be selected such that the modulation is linearly related
to the adjusted light intensity.
[0033] In certain embodiments, the methods can be implemented using
a microarray reader comprising the upstream selective light
modulator, and a light detector disposed downstream from the
microarray in a detection light path substantially at a conjugate
image plane of the sample, wherein the selective light modulator
and the light detector can be operably connected to at least one
controller containing computer-implemented programming that
controls transmissive characteristics of the upstream selective
light modulator and that compiles the modulated illumination light
and the adjusted light intensity, and wherein the controller
spatially varies the transmissive characteristics of the selective
light modulator to vary the modulated illumination light impinging
on the non-acceptable target spots of the microarray such that
light emanating from the non-acceptable target spots can be between
the threshold levels. The upstream selective light modulator can
comprise a digital micromirror device and the detector can comprise
a charge coupled device.
[0034] In other aspects, the present invention provides automated
methods of reading a microarray comprising, a) providing an initial
representation of a microarray comprising a plurality of target
spots illuminated by illumination light having a designated
intensity; b) determining from the initial representation whether
at least one of the target spots has an emanating light intensity
that can be not between selected upper and lower threshold values,
and designating at least one of such target spots a non-acceptable
target spot; and, c) modulating the emanating light intensity via
an automated downstream selective light modulator located in a
detection light path substantially at a conjugate image plane of
the sample to provide a modulated detection light comprising an
adjusted emanating light intensity that can be between the selected
upper and lower threshold values.
[0035] The methods can further comprise measuring the amount of
modulation of the detection light and measuring the modulated
detection light, then correlating the amount of modulation with the
modulated detection light to provide a measure of the actual signal
strength of the target spot. The methods can also further comprise
determining an amount of a probe located at the non-acceptable
target spot from the measure of the actual signal strength of the
target spot.
[0036] Such methods can be implemented using a microarray reader
comprising the selective light modulator, and a light detector
disposed in a detection light path substantially at a conjugate
image plane of the sample and downstream from the microarray and
the downstream selective light modulator, wherein the selective
light modulator and the light detector can be operably connected to
at least one controller containing computer-implemented programming
that controls transmissive characteristics of the downstream
selective light modulator and that compiles the modulated detection
light and the adjusted light intensity, and wherein the controller
selectively varies the transmissive characteristics of the
selective light modulator to vary the modulated detection light
impinging on the non-acceptable target spots of the microarray such
that light received at the detector can be between the threshold
levels.
[0037] In a further aspect, the present invention provides
microarray reader comprising an automated upstream selective light
modulator located upstream of a microarray in an illumination light
path substantially at a conjugate image plane of the sample, and a
light detector disposed downstream from the microarray in a
detection light path substantially at a conjugate image plane of
the sample, wherein the selective light modulator and the light
detector can be operably connected to at least one controller
containing computer-implemented programming that controls
transmissive characteristics of the upstream selective light
modulator and that compiles an amount of modulated illumination
light when the upstream selective light modulator can be modulated
and an adjusted light intensity emanating from a target spot on a
microarray receiving the modulated illumination light, and wherein
the controller selectively varies the transmissive characteristics
of the selective light modulator to vary the modulated illumination
light impinging on at least one non-acceptable target spot of the
microarray such that light emanating from the at least one
non-acceptable target spot can be between selected threshold
levels.
[0038] The controller can further comprise computer-implemented
programming that implements other aspects of the methods discussed
herein. For example, the programming can control measuring the
amount of modulation of the illumination light and control
measuring the adjusted light intensity, then correlate the amount
of modulation with the adjusted light intensity to provide a
measure of the actual signal strength of the target spot. The
programming can determine an amount of a probe located at the at
least one non-acceptable target spot from the measure of the actual
signal strength of the target spot.
[0039] In another aspect, the present invention provides microarray
reader comprising an automated downstream selective light modulator
located downstream of a microarray in a detection light path
substantially at a conjugate image plane of the sample, and a light
detector disposed in the detection light path substantially at a
conjugate image plane of the sample and downstream from the
downstream selective light modulator and the microarray, wherein
the downstream selective light modulator and the light detector can
be operably connected to at least one controller containing
computer-implemented programming that controls transmissive
characteristics of the downstream selective light modulator and
that compiles an amount of modulated detection light when the
downstream selective light modulator can be modulated and an
adjusted light intensity received by the detector, and wherein the
controller selectively varies the transmissive characteristics of
the downstream selective light modulator to vary the modulated
detection light emanating from at least one non-acceptable target
spot of the microarray such that light received at the detector
from the at least one non-acceptable target spot can be between
selected threshold levels.
[0040] As above, the controller can further comprise programming
that implements other features of the methods. The controller can
control measuring the amount of modulation of the detection light
and control measuring the adjusted light intensity, then correlate
the amount of modulation with the adjusted light intensity to
provide a measure of the actual signal strength of the target spot.
The controller can also determine an amount of a probe located at
the at least one non-acceptable target spot from the measure of the
actual signal strength of the target spot.
[0041] In still other aspects, the present invention includes
automated methods of reading a microarray comprising, a) providing
an initial representation of a microarray comprising a plurality of
target spots illuminated by illumination light having a designated
intensity; b) determining from the initial representation whether
at least one of the target spots has an emanating light intensity
that can be not between selected upper and lower threshold values,
and designating at least one of such target spots as a
non-acceptable target spot; c) selectively illuminating the
non-acceptable target spot via selectively transmitting light to
the microarray using a first automated upstream selective light
modulator located in an illumination light path substantially at a
conjugate image plane of the sample; and, d) modulating the
designated intensity of the illumination light via a second
automated upstream selective light modulator located in the
illumination light path substantially at a conjugate image plane of
an aperture diaphragm of the objective lens, to provide a modulated
illumination light and an adjusted target spot that emanates an
adjusted light intensity between the selected upper and lower
threshold values.
[0042] The methods can also modulate the designated intensity of
the illumination light via the first automated upstream selective
light modulator located in the illumination light path
substantially at the conjugate image plane of the sample, and can
determine an amount of a probe located at the adjusted target
spot.
[0043] In yet further aspects, the present invention comprises
automated methods of reading a microarray comprising, a) providing
an initial representation of a microarray comprising a plurality of
target spots illuminated by illumination light having a designated
intensity; b) determining from the initial representation whether
at least one of the target spots has an emanating light intensity
that can be not between selected upper and lower threshold values,
and designating at least one of such target spots as a
non-acceptable target spot; c) selectively detecting light from the
non-acceptable target spot via selectively transmitting light from
the microarray using a first automated downstream selective light
modulator located in a detection light path substantially at a
conjugate image plane of the sample; and, d) modulating the
emanating light intensity via a second automated downstream
selective light modulator located in a detection light path
substantially at a conjugate image plane of an aperture diaphragm
of the objective lens, to provide a modulated detection light
comprising an adjusted emanating light intensity between the
selected upper and lower threshold values.
[0044] The methods can further comprise also modulating the
emanating light intensity of the detection light via the first
automated downstream selective light modulator located in the
detection light path substantially at the conjugate image plane of
the sample.
[0045] In additional aspects, the present invention includes
microarray readers configured to implement such methods. For
example, the readers can comprise a first automated upstream
selective light modulator located upstream of a microarray in an
illumination light path substantially at a conjugate image plane of
the sample, a second automated upstream selective light modulator
located upstream of the microarray in the illumination light path
substantially at a conjugate image plane of an aperture diaphragm
of the objective lens, and a light detector disposed downstream
from the microarray in a detection light path substantially at a
conjugate image plane of the sample, wherein the first and second
selective light modulators and the light detector are operably
connected to a suitable controller.
[0046] Alternatively, the microarray reader can comprise a first
automated downstream selective light modulator located downstream
of a microarray in a detection light path substantially at a
conjugate image plane of the sample, a second automated downstream
selective light modulator located downstream of the microarray in
the illumination light path substantially at a conjugate image
plane of an aperture diaphragm of the objective lens, and a light
detector disposed in the detection light path substantially at a
conjugate image plane of the sample and downstream from the first
and second downstream selective light modulators and the
microarray, wherein the first and second selective light modulators
and the light detector are operably connected to a suitable
controller.
[0047] These and other aspects, features and embodiments of the
invention are set forth within this application, including the
following Detailed Description and attached drawings. In addition,
various references are set forth herein, including in the
Cross-Reference To Related Applications, that discuss in more
detail certain systems, apparatus, methods and other information;
all such references are incorporated herein by reference in their
entirety and for all their teachings and disclosures, regardless of
where the references may appear in this application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIGS. 1A and 1B provide a schematic view and an expanded
schematic view of a microarray reader according to one embodiment
of the present invention that comprises a selective light modulator
to replace the field diaphragm.
[0049] FIG. 2 provides a schematic view of a digital micromirror
device transmitting light as different functions of time.
[0050] FIGS. 3A and 3B provide a schematic view and an expanded
schematic view of a further embodiment of a microarray reader
according to the present invention in which an selective light
modulator is positioned upstream of, or before, a dichroic
mirror.
[0051] FIGS. 4A and 4B provide a schematic view of a microarray
reader according to another embodiment of the present invention
that uses two selective light modulators.
[0052] FIGS. 5A and 5B is a schematic view and an expanded
schematic view of a microarray reader according to still another
embodiment of the invention that comprises four digital micromirror
devices to control the light transmission characteristics of the
conjugate image plane of the sample and the conjugate image plane
of the aperture diaphragm of the objective lens.
[0053] FIG. 6 provides a schematic view of an area of a pixelated
light detector for use in the present invention showing an example
of the area of the detector illuminated by one of the mirrors of
the selective light modulator.
[0054] FIG. 7A provides a schematic view of a selective light
modulator in which multiple adjacent pixels are switched "on" to
define an illumination spot of a desired size.
[0055] FIG. 7B provides a schematic view of an selective light
modulator in which multiple adjacent pixels are rapidly switched on
and off such that when time averaged the mirrors are partially on
or partially off to define a Gaussian profile illumination
spot.
[0056] FIGS. 8A and 8B depict photographs of microarrays read with
and without modulation, respectively.
[0057] FIGS. 9A and 9B presents histograms of the measurement data
from FIGS. 8A and 8B.
DETAILED DESCRIPTION
[0058] The present invention enhances the dynamic range over which
microarray readers can measure target spots on a microarray, and
can improve the signal-to-noise ratio of such readers, by
dynamically modifying the illumination properties of the readers.
Modulating the amount of light directed at specific target spots,
such that dimmer target spots receive more excitation light and
overly bright target spots receive less light, increases or
decreases their measured brightness to more desirable ranges better
suited for the detector, and in some cases less likely to interfere
with readings of adjacent target spots. The dynamically modified
illumination properties can, if desired, be used instead of, or in
conjunction with, modifications to data sampling time, detector
sensitivity, and digitization accuracy (pulse counting or voltage
digitization). In some embodiments, the dynamically modified
illumination properties can increase the range of the microarray
reader by up to about 1000 times, and improve the signal-to-noise
ratio for target spots up to about 16 times.
[0059] Before discussing the Figures, it will be helpful to review
some definitions and to discuss various methods related to the
microarray readers of the present invention.
[0060] Definitions
[0061] A "selective light modulator" (SLM) is a device that is able
to selectively modulate light. In the present invention, selective
light modulators are disposed in the light path of a microarray
reader. The selective light modulator can be pixelated and comprise
an array of individual light transmission pixels, which is also
known as a spatial light modulator, which are a plurality of spots
that have transmissive characteristics such that they either
transmit or pass the light along the light path or block the light
and prevent it from continuing along the light path (for example,
by absorbing the light or by reflecting it out of the light path).
Such pixelated arrays are known, having also been referred to as a
multiple pattern aperture array, and can be formed, for example, by
an array of twisted-nematic or ferroelectric liquid crystal
devices, by a digital micromirror device (DMD), or by electrostatic
microshutters. See, U.S. Pat. No. 5,587,832; R. Vuelleumier, Novel
Electromechanical Microshutter Display Device, Proc. Eurodisplay
'84, Display Research Conference September 1984. Digital
micromirror devices can be obtained from Texas Instruments, Inc.,
Dallas, Tex., U.S.A. Other suitable devices include
microoptoelectromechanical system (MOEMs) with suitably high
contrast abilities (for example, 200-to-1 or 1000-to-1 contrast
ratios) between on-and-off states.
[0062] The "illumination light path" is the light path from a light
source to a microarray, while a "detection light path" is the light
path for light emanating from a microarray to a detector. Light
emanating from a microarray includes light that reflects from a
microarray, is transmitted through a microarray, or is created
within the microarray, for example, fluorescent light that is
created within a microarray pursuant to excitation with an
appropriate wavelength of light.
[0063] A "conjugate image plane of the sample" is a plane in either
the illumination light path or the detection light path wherein an
image of the microarray is recreated; and thus is a conjugate image
plane of the microarray. The light detector(s) is typically located
in one such site in the detection light path. The conjugate image
planes of the sample define locations that can control the size and
location of spots on the microarray that are illuminated and/or
detected (depending upon whether the conjugate plane is in the
illumination light path or the detection light path). The image
plane of the sample is the plane wherein the microarray is located,
although the image plane of the sample can be greater or smaller
than the size of the actual microarray, for example if either a
plurality of light paths are provided or if the illumination area
is greater or smaller than the size of the microarray.
[0064] A "conjugate image plane of an aperture diaphragm of the
objective lens" is a plane in either the illumination or detection
light path where an image of the aperture diaphragm of the
objective lens is recreated. Typically, this image plane also
contains a recreation of the image of the light source, which in
the present invention can be any light source such as a white
light, an arc lamp or a laser. The conjugate image planes of the
aperture diaphragm of the objective lens define locations that
control the angle of illumination light that is ultimately impinged
on a microarray, as well as the angle of detection light that
emanates from a microarray (the "angle of illumination" and "angle
of detection" refer to the angle of the light that is either
impinging upon or emanating from a microarray).
[0065] A "controller" is a device that is capable of controlling a
selective light modulator, a detector or other elements of the
apparatus and methods of the present invention. For example, the
controller can control the light communication characteristics of a
selective light modulator, control the on/off status of pixels of a
pixelated selective light modulator or light detector (such as a
charge coupled device (CCD) or charge injection device (CID)),
and/or compile data from the selective light modulator or the
detector, including using such data to make or reconstruct images
of a microarray or as feedback to control an upstream selective
light modulator. Typically, a controller is a computer or other
device comprising a central processing unit (CPU) or other
logic-implementation device, for example a stand alone computer
such as a desk top or laptop computer, a computer with peripherals,
a local or internet network, etc. Controllers are well known in the
art and selection of a desirable controller for a particular aspect
of the present invention is within the scope of the art in view of
the present disclosure.
[0066] "Upstream" and "downstream" are used in their traditional
sense wherein upstream indicates that a given device is closer to a
light source, while downstream indicates that a given object is
farther away from a light source.
[0067] The scope of the present invention includes both means plus
function and step plus function concepts. However, the terms set
forth in this application are not to be interpreted in the claims
as indicating a "means plus function" relationship unless the word
"means" is specifically recited in a claim, and are to be
interpreted in the claims as indicating a "means plus function"
relationship where the word "means" is specifically recited in a
claim. Similarly, the terms set forth in this application are not
to be interpreted in method or process claims as indicating a "step
plus function" relationship unless the word "step" is specifically
recited in the claims, and are to be interpreted in the claims as
indicating a "step plus function" relationship where the word
"step" is specifically recited in a claim.
[0068] Other terms and phrases in this application are defined in
accordance with the above definitions, and in other portions of
this application.
[0069] Turning to some methods of the present invention, the
methods comprise dynamically modifying the illumination properties
of the reader. In certain embodiments, this is effected by
illuminating a microarray to provide an initial representation of
the microarray, which can be an image, precompiled map of expected
data (for example from previous experiments or as intentionally
designed), or other representation of the photonic or light
intensity emanating from the microarray. Such initial
representation of desired spots on the microarray is measured, and
then for any spot falling outside of desired parameters, i.e.,
non-acceptable target spots have greater than or less than certain
upper and lower threshold levels (maxima and minima) of emanating
light, respectively, increasing or decreasing the amount of
illumination light directed to such non-acceptable target spot by
selectively modulating the light via a spatial light modulator
(SLM). The amount of modulation is then correlated with the amount
of light detected by the detector; the intensity of the
illumination light and the emanating light intensity from the given
target spot on the microarray are considered together. The system
then provides a reading of the actual light intensity emanating
from the target spot, taking into account both the intensity of the
illumination light and the intensity of the detected light. Because
the dynamic range of the spatial light modulator and the detector
are additive (or multiplicative, depending upon the method of
representation), the system has a substantially increased dynamic
range compared to systems that do not modulate the illumination
light. The following paragraphs typically discuss the methods and
systems for exemplary purposes using a digital micromirror
device(s) as the SLM(s) and CCD(s) as the detector(s), but other
structures are also suitable.
[0070] In some embodiments of effecting such methods, before
readings are taken (or while readings are being taken, as desired)
specific mirrors of the DMD or other SLM are correlated with
specific pixels or areas of the CCD detector or other pixelated
detector. This can be done using any desired, appropriate
calibration technique, for example techniques similar to those used
to map DMD mirrors (or the light controllers of other SLMs, such as
MOEMs) to fiber cores discussed in U.S. patent application Ser. No.
09/738,257, entitled Methods And Apparatus For Imaging Using A
Light Guide Bundle And A Spatial light modulator, filed Dec. 14,
2000. Once mapping has been done for a particular set up, typically
it does not need to be repeated until either the DMD or CCD is
moved relative to the other.
[0071] In some embodiments, the DMD is located at or near a
conjugate image plane of the sample in either an epi-illumination
or transmission microarray reader configuration; other
configurations are also possible. For each new microarray area or
target spot that is imaged, an initial image is acquired using a
pre-set exposure time. The bright areas and the dim areas (spots)
in the image are then noted. Next, the DMD mirrors corresponding to
the very bright target spots are set to illuminate less intensely,
and the DMD mirrors corresponding to the dim target spots are set
to illuminate more intensely, etc. A second image is then obtained,
which image has more uniform target spot image intensities. The
actual amount of target material (e.g., labeled probe) located
within a target spot can be determined by the emanating light
intensity of the target spot in the image and by the intensity
(e.g., dwell time) of the illumination directed to each target
spot.
[0072] The dynamic range of the DMD-CCD system is that of the DMD
plus that of the CCD. Thus, if the DMD can provide 1000 different
illumination levels and the CCD has 1000 distinct grey levels of
detection, then (ignoring photon noise and other possible
artifacts) the system has a dynamic range (within a single
microarray) of 1,000,000 or 10.sup.6, a 1,000 fold increase.
[0073] It is also possible to use the combination of the DMD and
CCD dynamic ranges to provide superior signal-to-noise
characteristics. Briefly, the number of photons detected (counted)
at lower grey level values can have a worse signal-to-noise ratio
because of photostatistics or other hindering influences. For
example, if a maximum grey level of 10,000 corresponds to a full
well capacity of 10,000 electrons, then signals with a value of
10,000 have a noise level of 100 (signal-to-noise of 100-to-1)
whereas signals with a grey level value of 100 have a noise value
of 10 (signal-to-noise of 10-to-1). So target spots (in a cDNA
microarray, for example) that are about 100 times different in
their emanating light intensity (the strong signal spots having a
signal level of 10,000 and the weak spots having a signal level of
100) vary significantly in their signal-to-noise ratios (by about
10 fold in this example).
[0074] In accordance with the present invention, the illumination
light intensity on the target spots can be modulated such that the
grey levels of each target spot at the detector are about 10,000 by
illuminating the weak signal spot with 100 times more intense light
than the strong signal spot. This results in the two target spots
having closer, up to substantially the same, signal-to-noise values
or ratios. The overall signal strength of each target spot is
provided by the intensity (or duration) of the illumination signal
in combination with the detected light, so an enhanced, and more
consistent signal-to-noise ratio can be achieved simultaneously
with improved dynamic range.
[0075] The methods and system can be set up such that for each
frame or image of a microarray to be measured (target spots read)
either a priori information about target spot strength and location
from a database or an initial image of the frame can be used to
modulate the intensity of the illumination light to get uniformly
high grey level intensity images for each target spot. From the
individual spot illumination intensity or duration, the strength of
each spot can be calculated. In addition, the results can be
further improved by either measuring the photobleaching
characteristics of each target spot or fluorophore by using a
sequence of images or by using previously acquired photobleaching
characteristic information to correct the measured spot
strength.
[0076] Ignoring photobleaching, the relationship between the target
spot strength (i.e., emanating light intensity of the target spot;
SS(x,y)), illumination intensity or duration (II(x,y)), and
detected CCD signal (CCDS(x,y)) can be represented as:
SS(x,y)=K*CCDS(x,y)/II(x,y)
[0077] where K is a constant for the system.
[0078] Including a photobleaching function, PB(II(x,y),fluoro),
which is a function of illumination energy/intensity and the
fluorophore being excited, the relationship becomes:
SS(x,y)=K*PB(II(x,y),fluoro)*CCDS(x,y)/II(x,y)
[0079] Since, in some circumstances, the photobleaching process can
be a function of other factors (oxygen, chemical environment, etc.)
which may vary from location to location within the microarray,
PB(II(x,y), fluoro) can, if desired, be determined on the fly and
incorporate an addition spatial variation term
PB(II(x,y),fluoro,x,y). To determine the photobleaching behavior on
a location-by-location basis, a sequence of images (two or more)
can be taken, and the spatial photobleaching function can be
determined for the specific microarray frame. Various forms of
photobleaching functions can be used (single exponential, double
exponential, multi exponential, etc.), and the multiple image data
used to generate the model parameters which give the suitable fit
to the observed data. This spatially dependent photobleaching
function can then be used to determine the signal strength in the
microarray.
SS(x,y)=K*PB(II(x,y),fluoro,x,y)*CCDS(x,y)/II(x,y)
[0080] In addition to the above mentioned benefits, the equations
can improve measurement of target spot strength by accounting for
differential photobleaching of different fluorophores.
[0081] The more uniform target spot intensities described above and
elsewhere herein simplify glare or scatter corrections, if any, for
the measured image, and can correct, for example, for the nonlinear
or other undesired behavior of the imaging optics. An image which
has had glare and scatter removed from the image data produces more
accurate spot intensity measures, for example because the spots are
more accurate and because the background values used to determine
spot intensity values are more representative of what actually
occurred in the microarray.
The Figures
[0082] Exemplary microscopic systems suitable for use with the
present invention are depicted in FIGS. 1-5B. The systems can be
confocal, wide field, or otherwise configured as desired. Other
systems can also be used.
[0083] FIGS. 1A and 1B depict a schematic drawing of a microarray
reader 2 comprising a selective light modulator in an upstream
conjugate image plane of the sample. Microarray reader 2 comprises
a light source 4 that emits a plurality of illumination light rays,
such as excitation light rays, along illumination light path 3
toward the microarray 20. The light rays have been divided into
first light rays 6, second light rays 8 and third light rays 10.
The light rays first pass through a filter 36, then reflect off a
dichroic mirror 38 (the dichroic mirror 38 and filter 36 are
maintained in a dichroic mirror and filter block set 28) and
through a projection lens 30, followed by reflection off a simple
mirror 32 onto a selective light modulator, which in the figure is
a digital micromirror device 34. As depicted in the figure, all of
the individual light transmission pixels (i.e., micromirrors in the
figure) are on, and thus all of light rays 6, 8, 10 are transmitted
to objective lens set 22 and microarray 20. If one or more of the
individual light transmission pixels were turned off, the light
rays would be directed to a second or third location, for example a
beam stop or additional detector 37. The light is then reflected or
fluoresced off microarray 20 and back through objective lens 22,
off digital micromirror device 34 and simple mirror 32 and then
transmitted through projection lens 30. The light then continues
past dichroic mirror 38, filter 36 and ultimately to light detector
26. The light is transmitted from the microarray to the light
detector 26 along detection light path 5.
[0084] In FIGS. 1A and 1B, digital micromirror device 34 is placed
in a conjugate image plane of the sample in each of the
illumination light path 3 and the detection light path 5. Light
detector 26 can be any desired light detector, for example, a
detector comprising a charged coupled device (CCD), a charged
injection device (CID), a complementary metal-oxide semi-conductor
(CMOS), or a video camera. If desired, it is possible to use a
plurality of different light detectors either in series or in an
adjacent relationship or in any other desired relationship. In some
typical embodiments, the light detector is a CCD or a CID or other
light detector that comprises as array of individual detection
pixels, which indicates a plurality of spots, typically on the same
order of the same size as the pixels in the selective light
modulator.
[0085] In some embodiments, the detection array of individual
detection pixels in light detector 26 corresponds to and is aligned
with the illumination array of individual light transmission pixels
in the selective light modulator. Accordingly, the detection array
can have an equivalent number of pixels, each of which is aligned
with the pixels of the selective light modulator array, or groups
of such pixels are aligned with each other. In certain embodiments,
this alignment can be effected by using a single digital
micromirror device at a desired conjugate image plane in both the
illumination light path and the detection light path, for example
as depicted in FIGS. 1A and 1B.
[0086] FIGS. 1A and 1B depict an epi-fluorescence microarray
reader, which means that the light is incident on the microarray
from above, but it could also represent a transmission microarray
reader, which would mean the light would be incident from below and
transmit through the microarray, if a separate second selective
light modulator were used in the detection light path (or if
appropriate mirrors or other devices were to direct the detection
light path back to a single digital micromirror device).
[0087] FIG. 2 depicts a schematic representation of a microarray
reader comprising a selective light modulator in a conjugate image
plane of the sample, and illustrates how a digital micromirror
device can produce grey-scale intensities. In particular, digital
micromirror device 34 reflects a plurality of light beams along
illumination path 3 to projection lens 30 and into image plane 40.
In a central column of the digital micromirror device 34, each of
the digital micromirrors has a different percentage of time in
which the mirror is on instead of off. For example, the top most
micromirror in the figure is on 100% of the time, while the bottom
most micromirror in the column in the figure is off 100% of the
time, while the four mirrors between the two have an on/off status
that is between 100% on and 100% off. Thus, each of the light rays
from the central column have a different video field time 50, which
video field time corresponds to the amount of on and off time for
the particular micromirror.
[0088] FIGS. 3A and 3B depict a schematic view of a microarray
reader that is similar to the microarray reader set forth in FIGS.
1A and 1B, except that the selective light modulator 34 is disposed
solely in the illumination light path and not in the illumination
light path 3.
[0089] Typically, the on/off pixel pattern(s), or patterns of other
light modulation effectors, in the selective light modulator(s) is
effected via operably connecting the selective light modulator to a
controller, such as a PC computer, that individually controls each
of the individual light transmission pixels or other light
modulation effector. Where the selective light modulator is a DMD
or other pixelated device, the controller can control a single
mirror as a single pixel or a plurality of mirrors as a single
pixel. For example, each individual light transmission pixel can be
a grouping of immediately adjacent mirrors, such as set forth in
FIGS. 7A and 7B. In particular, FIGS. 7A and 7B schematically
depict two different embodiments for illumination comprising the
use of adjacent mirrors as a single pixel. In FIG. 7a, a plurality
of individual micromirrors of the selective light modulator are
turned on as a group. FIG. 7B depicts a similar illumination spot
except that different micromirrors (or microshutters or other
selected pixel components) have different on/off status and thus
provide a Gaussian illumination profile; other illumination
profiles are also possible.
[0090] In one embodiment for correcting or varying the intensity of
the light impinging on the selected target spots of the microarray
the light from light source 4 that contacts "off" pixels in the
array of the selective light modulator such as digital micromirror
device 34 is transmitted to a light detector 37 in FIGS. 1A and 1B.
The light detector 37 can receive light directly from the selective
light modulator without first going through, or reflecting off, the
microarray. The light may or may not go through intervening lenses,
filters, etc., between the selective light modulator and the light
detector 37. The light detector 37 is typically located at a
conjugate image plane of the sample, or in the image plane of the
sample itself. When the selective light modulator 34 has all pixels
turned "off" all of the light from the light source is transmitted
to the detector 37. The detector 37 can then differentiate
different levels of intensity within the light emanating from the
light source 4, and then to correct for variant intensities via
rapid alternating between on and off status to provide a
substantially uniform light to microarray 20. This enhances the
ability of the system to obtain accurate initial readings from the
microarray 20 before the light intensity modulations discussed
elsewhere herein. This also permit the microarray reader to have a
back up or different approach to measuring the illumination light
impinging on the microarray; instead of, or in addition to,
figuring the amount of illumination light by measuring the amount
of on/off time for the relevant pixels in the selective light
modulator, the intensity of the modulated light can be directly
measured at the light detector 37, for example by switching the SLM
to shine the same portion of light that would go to the microarray
to the detector, or by subtracting from the total illumination
light intensity the re-directed light that would have gone to the
microarray but instead is directed to the detector 37.
[0091] In a related embodiment, the light detector and controller
can in some embodiments determine the light intensity
characteristics of a microarray by detecting the intensity of the
light impinging on a detector downstream from the microarray, then
modifying the on/off status of the corresponding pixels in the
illumination array until a substantially uniform intensity of light
is transmitted to the pixels of the detector array, and then
determining the light intensity characteristics of the microarray
by determining the amount of time that individual pixels in the
illumination light array in the selective light modulator are on or
off, or otherwise reducing the amount of light transmitted to the
microarray.
[0092] FIG. 6 schematically depicts an embodiment wherein the
central detection pixel 56 is more heavily illuminated than
adjacent or surrounding pixels 58, but due to the characteristics
of the microarray the surrounding pixels are, in fact, illuminated
even though only central pixel 56 was directly aligned with the on
illumination pixel in the illumination array of the selective light
modulator. Thus, in an embodiment that is useful for confocal
microscopy and other forms of microarray readers, the microarray
reader comprises a controller that contains computer implemented
programming that causes the light detector to detect light
impinging on a central detection pixel that is aligned with a
corresponding individual light transmission pixel of the selective
light modulator in the illumination light path that is on and also
to detect light impinging on at least one pixel adjacent to the
central detection pixel, typically all adjacent pixels. The
controller also contains computer-implemented programming that
compiles the data provided by the adjacent detection pixel(s) and
combines it with the data provided by the central detection pixel
to enhance the information provided to and by the microarray
reader. For example, such combining of the data can enhance the
rejection of the out-of-focus information of the microarray reader
when such rejection is compared to the focus that is attained
without the data from the adjacent detector(s). Alternatively, the
information from the adjacent pixels can provide data about the
light scattering and/or absorption or other characteristics of the
microarray. Alternatively, if desired, the detector can be set such
that the detector and/or controller does not detect information
from the central detector pixel that directly corresponds to the on
illumination pixel but rather only collects information from the
adjacent pixel(s).
[0093] FIGS. 4A and 4B depict one example of a microarray reader
that is similar to the microarray readers depicted in FIGS. 1 and
3, except that the microarray reader in FIGS. 4A and 4B comprise a
first digital micromirror device in a conjugate image plane of the
sample that is upstream from the microarray and a second, separate
digital micromirror device 34b that is located in a conjugate image
plane of the sample that is disposed downstream from the
microarray. Thus, in FIGS. 4A and 4B, light is emitted by light
source 4 through projection lens 30a to reflect off simple mirror
32a and then selective light modulator 34a. Light that is
transmitted along the illumination light path by the selective
light modulator 34a is then reflected off dichroic mirror 38,
through objective lenses 22 onto microarray 20, where the light is
reflected back through objective lenses 22, then through dichroic
mirror 38 and onto downstream selective light modulator 34b. Light
that is passed by selective light modulator 34b continues along the
detection path to simple mirror 32b, then through projection lens
30b to light detector 26. One advantage of the microarray reader
depicted in FIGS. 4A and 4B is that, because there are two separate
selective light modulators, the two selective light modulators need
not have identical on/off status for the light transmission pixels
therein. Similar to many other embodiments herein, the embodiment
in FIGS. 4A and 4B can be used both with pixelated and
non-pixelated detectors such as a photomultiplier tube (PMT), video
camera, or other device. In addition, if desired, the detection
aperture in the downstream selective light modulator in the
detection light path can be dynamically varied in the same manner
as described earlier for selective light modulators disposed in the
illumination light path.
[0094] The modulation of the light striking the light detector 26
can also be effected by modulating the transmission characteristics
of the second digital micromirror device 34b that is disposed
downstream from the microarray. For example, if the light emanating
from the microarray is too bright, then a desired portion of the
light can be removed from the detection light path by the second
digital micromirror device 34b, and then the on/off status of the
second digital micromirror device 34b can be considered in
conjunction with the light detected by the detector, to provide the
actual spot signal strength, similar to the manner in which the
spot signal strength is determined using an upstream selective
light modulator.
[0095] FIGS. 5A and 5B schematically depict one embodiment of a
microarray reader suitable for 3-D imaging of the microarray.
Briefly, FIGS. 5A and 5B comprise four selective light modulators,
two in the illumination light path and two in the detection light
path, one in each light path located in the conjugate image plane
of the sample and one in each light path located in the conjugate
image plane of the aperture diaphragm of the objective lens.
Accordingly, in the Figure, light source 4 emits light to simple
mirror 32a, which reflects light to a first selective light
modulator 34a, which is located in an upstream conjugate image
plane of the aperture diaphragm of the objective lens. Selective
light modulator 34a transmits a desired portion of light along
illumination light path 3 to projection lens 30a, after which it is
reflected off simple mirror 32b and transmitted to a second
selective light modulator 34b, which selective light modulator is
located in an upstream conjugate image plane of the sample. The
second selective light modulator 34b then transmits desired light
through condensor lenses 16 to microarray 20 where the light is
transmitted through the microarray 20, through objective lenses 22
and onto a third selective light modulator 34c, which is located in
a downstream conjugate image plane of the sample.
[0096] FIGS. 5A and 5B also depict an adjustable iris aperture
diaphragm (condenser) 18 that can be disposed between upstream and
downstream condenser lenses 16. The light then contacts, or
impinges upon, microarray 20 and then proceeds to pass through
objective lenses 22, which objective lenses can comprise an
aperture diaphragm (objective) 24 space between the objective
lenses 22. Diaphragms such as diaphragms 16, 24 can be used with
other embodiments of the microarray readers discussed herein if
desired. Light that is desired to be transmitted to the light
detector 26 is then transmitted by the third selective light
modulator 34c to simple mirror 32c where it is reflected through
projection lens 30b and onto a fourth selective light modulator
34d, which modulator is located in a downstream conjugate image
plane of the aperture diaphragm of the objective lens. The fourth
selective light modulator then transmits desired light to simple
mirror 32d which reflects the light to light detector 26.
[0097] Using the exemplary system of FIGS. 5A and 5B, it is
possible to spatially combine various features for selective light
modulators located in one or another of the conjugate image planes.
For example, combining confocal microscopy with illumination at a
variety of angles provides for 3-D confocal transmission and
reflectance microscopy. Further, because of the rapid switching
time that is possible using the selective light modulators, it is
possible to see such 3-D confocal image in real time. Additionally,
due to the ability to calculate, and account for, out-of-focus
information as discussed herein, such information can be limited
and controlled, thereby simplifying the reconstruction task in the
making of the 3-D image. Examples of other systems comprising
selective light modulators in the conjugate image plane of the
aperture diaphragm of the objective lens are discussed in U.S.
patent application Ser. No. 09/179,185, filed Oct. 27, 1998.
[0098] Configurations such as those depicted in FIGS. 5A and 5B
where the microarray reader comprises a selective light modulator
in the conjugate image plane of the aperture diaphragm of the
objective lens provide additional approaches to modulating the
detected signal strength emanating from a given spot(s) on a
microarray. Briefly, the selective light modulator in the conjugate
image plane of the sample can be used to selectively illuminate or
detect a given spot, typically a spot too dim or too bright, then
the selective light modulator in the conjugate image plane of the
aperture diaphragm of the objective lens can modulate the amount of
light to or from the spot by directing undesired light along a
different light path, to a beam stop or detector other desired
light receptacle.
[0099] In alternative embodiments of the invention that account for
too dim or too bright spots on microarrays, the dynamic range of a
microarray reader can be improved by using differential, known
sensitivity settings for different spots. This can be accomplished,
for example, by varying the gain settings on a PMT for a scanning
spot microarray reader, or by providing different binning options
for different image locations on the CCD of a CCD based microarray
reader. It is also possible to acquire more than one image with
each image having different, known exposure settings. For example,
for too dim spots a long exposure time is used and for too bright
spots a short exposure time is used. The different images can then
be correlated with their respective exposure times and the actual
signal strength determined. For a scanning spot microarray reader
it is also possible, after the first image has been acquired, to
intensity modulate the scanning illumination beam such that bright
spots in the images receive a known less amount of light and that
the dim spots receive a known larger amount of light.
[0100] Another advantage of the present invention is that it
permits easily performed time-delayed fluorescence microscopy. This
can be accomplished by turning on desired illumination pixels in
the selective light modulators in the illumination light path and
then turning off corresponding pixels in the selective light
modulators (or detector) in the detection light path. After enough
time has passed to induce fluorescence in the microarray, which
fluorescence can be autofluorescence or fluorescence due to
materials, such as dyes, added to the microarray, the selective
light modulators in the illumination light path are turned off and
short time later the detection pixels of the detector, or the light
transmission pixels of selective light modulators disposed in the
detection path, are turned on. Examples of suitable timings for
such situations are discussed in U.S. patent application Ser. No.
09/179,185, filed Oct. 27, 1998. Such microscopy can be performed
both in confocal and wide field modes, or otherwise as desired.
EXAMPLE
[0101] A microarray reader similar to the microarray reader
depicted in FIGS. 3A and 3B was employed using a DMD (Texas
Instruments), conventional microscope optics and illumination
design, and an uncooled CCD (KAF-1400 Kodak) with double-correlated
12-bit sampling. Individual DMD mirrors in the illumination path
were mapped to CCD camera pixels. A series of gray scale target
images of differing intensity were transferred to the DMD to
determine the size of the inverse gamma function, which was
automatically applied by the DMD formatting electronics to images
displayed by the DMD. Using constant illumination, images of a
small part of an old and faded (and likely photobleached) spotted
microarray were acquired, FIG. 8A. This image was then inverted,
the gray levels remapped using an approximate gamma function, the
spatial pixel distribution remapped to correspond to the
appropriate DMD mirrors, applied to the DMD and a new image, FIG.
8B, was acquired. In this fashion the dim target spots in Fig. 8A
received proportionally more illumination than did the bright
target spots in Fig. 8A. However, because the light source (an arc
lamp) used was not intensity adjustable, the exposure time for Fig.
8B was substantially longer than for Fig. 8A. To calculate the
data, the uniform photon count for FIG. 8B was divided by the
illumination intensity applied to the DMD before the inverse gamma
correction.
[0102] As a result of this, in FIG. 8A (non-corrected illumination)
the maximum measured density (16 bit image) was 63242. For data
measured from FIG. 8B (modulated illumination) the maximum measured
intensity was 62976.
[0103] In FIG. 8A the measured intensity for area 1 was 1290 with a
SD of 155 and a signal-to-noise ratio of 8.2. For data measured
from area 1 in FIG. 8B the measured intensity was 1314 with a SD of
143 and a signal-to-noise ratio of 9.1.
[0104] In FIG. 8A the measured intensity for the background in area
2 was 380 with a SD of 159 and a signal-to-noise ratio of 2.4. For
FIG. 8B the measured intensity for the background in area 2 was 409
with a SD of 99 and a signal-to-noise ratio of 4.1.
[0105] FIGS. 9A and 9B, presents histograms of the measurement data
from FIGS. 8A and 8B. The first histogram shows the result of 12
bit sampling in a 16 bit image, which can be seen in the discrete
nature of the first histogram (since the four most least
significant bits are zero), whereas the second histogram represents
true 16 bit data. Nevertheless, the noise background of the second
histogram is less than the in the in the first histogram, as
indicated by the narrower background peak.
[0106] Based on these result, noise (uncertainty) in the background
of FIG. 8A is 60% greater than that in FIG. 8B, and the respective
signal-to-noise ratios of the dim and the bright areas are more
similar in FIG. 8B (9.2 for area 1 (bright area) and 4.1 for
background) than in FIG. 8A (8.2 for area 1 (bright area) and 2.4
for background).
[0107] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been discussed herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention includes such modifications as well as
all permutations and combinations of the subject matter set forth
herein and is not limited except as by the appended claims.
* * * * *